Stroma-Derived Exosomes Mediate Oncogenesis in Multiple Myeloma

Bone marrow (BM)-derived mesenchymal stem cells (MSCs) support multiple myeloma (MM) cell growth, but little is know about the putative mechanisms that may regulate the interaction between clonal MM plasma cells and the surrounding BM milieu. It is known that cell-to-cell communication is partially mediated by exosomes. We therefore characterized the role BM-MSCs-derived exosomes as key regulators of MM pathogenesis in vivo and in vitro.

MSCs were collected from BM of either healthy subjects or relapsed/refractory MM patients. MM cell lines (MM.1S; RPMI.8226) and normal BM stromal cell line (HS-5) were used. Purity of BM-MSCs was evaluated by flow cytometry (CD34⁻,14⁻, 45⁻, 19⁻, 138⁻; CD73⁺, 90⁺, 105⁺, 106⁺). Exosomes were collected from conditioned medium of either normal and MM BM-MSCs, or HS-5 cells; and studied using electron microscopy, immunogold labeling, and western blot for the detection of CD63 and CD81. Transfer of PKH67-fluorescently labeled exosomes to MM cells was evaluated by confocal microscopy and fluorescence plate reader. Transfer of murine-derived miRNA-containing exosomes into human MM cell lines was evaluated by qRT-PCR (exosomes were collected from BM-MSCs of C57BL/6 miRNA-15a/16-1^−/− or C57BL/6 mice). miRNA expression profiling was obtained from normal (n=4) and MM (n=9) BM-MSCs-derived exosomes (TaqMan human miRNA profiling). Normal and MM BM-MSCs-derived exosomes were loaded into tissue-engineered bones (TEB) with MM.1S-GFP+/Luc+ cells: MM cell homing and MM tumor growth has been tested in vivo by using in vivo confocal microscopy and bioluminescence (BLI) imaging, respectively. Normal and MM BM-MSCs, as well as HS-5 cells, were transfected with either anti- or pre-miRNA-15a or scramble probe; and evaluated for their ability to modulate MM cell proliferation and adhesion in vitro.

Primary normal and MM BM-MSCs release CD63+/CD81+ exosomes, as confirmed by electron microscopy, immunogold labeling, and western blot. BM-MSCs exosomes are transferred into MM cells, as shown by confocal microscopy. This transfer was further confirmed in human MM cell lines incubated with murine (C57BL/6 miRNA-15a/16-1^−/− and wild type) BM-MSCs-derived exosomes: qRT-PCR showed presence of murine miRNAs in human MM cell lines. The impact of normal and MM BM-MSCs-derived exosomes on MM cell behavior in vivo was next evaluated. MM cells co-cultured with exosomes derived from MM BM-MSCs induced rapid tumor growth at the site of the TEB scaffold, as well as rapid dissemination in the BM niches. In contrast, MM cells co-cultured with exosomes derived from normal BM-MSCs led to minimal tumor growth and minimal dissemination at distant BM niches. These results indicate that MM BM-MSCs-derived exosomes contribute to tumor growth and dissemination of MM. To further explore the mechanisms by which exosomes induce tumor growth, we performed miRNA expression profiling on exosomes isolated from both normal and MM BM-MSCs: supervised hierarchical clustering analysis showed increased expression of 24 miRNAs and reduced expression of 3 miRNAs in MM BM-MSCs-derived exosomes versus normal (1.5 fold change; P<0.05). Notably, we found that miRNA15a is significantly lower in exosomes derived from BM-MSCs of MM patients. We previously showed that miRNA15a shows lower expression in primary MM cells. We therefore sought to examine whether genetic transfer of miRNAs or lack of transfer of tumor suppressor miRNAs (such as miRNA15a) can lead to the significant change in tumor growth and dissemination in MM that we observed in vivo. We therefore transfected HS-5 stromal cells and primary normal BM-MSCs with pre-miRNA15a, and found that by over-expressing miRNA-15a in BM-MSCs inhibited MM cell proliferation and adhesion to fibronectin. Next MM cells were cultured in presence of BM-MSCs isolated from either C57BL/6 mice or C57BL/6 miRNA15a/16^−/: miRNA15a-deficient BM-MSCs significantly induced MM cell proliferation (P<0.05). Moreover, exosomes isolated from HS-5 pre-miRNA15a-transfected cells both inhibited MM cell proliferation and reduced their adhesion properties.

These findings demonstrate the existence of exosome-driven interactions between the BM milieu and MM cells, and suggest that exosomes might constitute a novel mechanism for intercellular transfer of genetic information in the form of miRNAs in clonal plasma cell disorders, such as MM.

Roccaro:Roche: Advisory Board. Anderson:Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Ghobrial:Novartis: Advisory Board; Celgene: Advisory Board; Millennium: Advisory Board; Noxxon: Advisory board; Millennium: Research Funding; Bristol-Myers Squibb: Research Funding.